Therapeutic method for activating factor VII

ABSTRACT

Patients having a clotting factor defect can be successfully treated by the administration of a composition containing effective hemostatic amounts of factor VIIa. Preferably, the factor VIIa is substantially free of factors IXa and Xa. Procedures for preparing the above therapeutic compositions are also described. A method involving the activation of factor VII is set forth.

This is a division of application Ser. No. 277,469, filed June 25, 1981,now U.S. Pat. No. 4,382,083.

BACKGROUND OF THE INVENTION

This invention relates to methods for treating patients havingdeficiencies or inhibitors of blood clotting factors, particularlyfactor VIII (antihemophilic factor, AHF).

Blood coagulation is an exceedingly complex process. The interaction ofvarious blood components which eventually gives rise to a fibrin clothas been compared to a cascade of steps, each of which is dependent uponand regulated by preceding and following steps. Generally, the bloodcomponents which take part in the coagulation cascade are eitherproenzymes or enzyme modulators. The proenzymes are enzymaticallyinactive proteins which are converted to proteolytic enzymes by theaction of an "activator", generally another proteolytic enzyme producedat an earlier stage in the coagulation cascade. Coagulation factorswhich have undergone such a conversion are hereafter defined asactivated factors, and designated by the lower case postscript "a" whilethe proenzymes are referred to as precursor clotting factors.

The enzyme modulators are principally cofactors such as calcium ions ornonenzyme proteins and most are essential if the enzymes are to exhibitany catalytic activity at all. Such modulators are to be distinguishedfrom enzyme substrates. Substrates are compounds which are covalentlymodified by an enzyme while modulators or cofactors merely bind to theenzyme without undergoing a change in structure.

Proenzymes are key components in the blood coagulation cascade. Theyconstitute a reservoir to supply the needs of the clotting cascade,being activated to the enzymatically functional form as required. Theextent of activation and the activity of the enzymes are controlled bythe modulators.

Factor VII is one of these proenzymes. Factor VII participates in theextrinsic pathway of blood coagulation by converting factor X to Xa inthe presence of tissue factor. Factor Xa in turn then convertsprothrombin to thrombin in the presence of the cofactors factor V,calcium ions and phospholipid. Recent evidence indicates that factorVIIa may participate in the intrinsic clotting pathway as well.

Bovine factor VII has been isolated and characterized (Kisiel and Davie,"Biochemistry" 14 (22): 4928-4934 [1975]). The factor VII purified bythese authors was homogeneous when examined by gel electrophoresis inthe presence of sodium dodecyl sulfate, exhibited less than 0.001 Orthounits of prothrombin/ml and undetectable levels of factors IX and X whenassayed at 0.5 mg protein/ml concentration. Since Factor VII activitylosses are high at low protein concentrations, 1 mg/ml of bovine serumalbumin was included in factor VII diluents as a stabilizer.

Bovine factor VII is a single-chain glycoprotein of approximately 50,000daltons. It is converted to a two chain disulfide-linked protein byfactor Xa in the presence of calcium ions and phospholipids, or bythrombin or factor XIIa without cofactors. An increase in factor VIIactivity of about 10 to 100 fold as measured in a one-stage coagulationassay is associated with the conversion to the two chain form.

Human factor VII has also been purified and characterized (Broze andMajerus, "J. Biol. Chem." 1242-1247 [1780]. This method yields factorVII which, like the bovine product obtained by Kisiel and Davie, waswithout detectable activity by coagulation assay and did not containdetectable factors X, IX or II. The final preparation contained 2.3units of factor VII/μg of protein. The human factor VII is 55-85%homologous with the bovine protein and shows many other characteristicsin common with bovine factor VII. Like bovine factor VIIa, it isconverted to a two chain form upon activation, apparently viaproteolytic cleavage. The activity increase upon activation to the twochain from is 20-25 fold. This two chain form will be synonymoushereinafter with factor VIIa.

Factor VIIa previously has not been employed to the applicant'sknowledge to treat patients with clotting inhibitors or deficiences.However, factor VIIa is known to be present in activated prothrombincomplex (PCC), a complex mixture of activated and unactivated clottingenzymes used to treat patients having inhibitors of factor VIII.

The therapeutically effective substances in activated prothrombincomplexes have been speculated to be one or more of factors IXa, Xa,XIa, XIIa, VIIa, Xa dimer, Xa-prothrombin intermediate binary complex,"large quantities" of factor VII, a "modified form" of factor VII,factor VIII inhibitor bypassing activity (FEIBA), platelet factor Xactivator and, to a small degree, thrombin. The overriding themeespoused by the art, however, is that an incomplete understanding existsof the in vivo mechanism of action for activated PCC, and therefore theart has been able to only speculate as to the effective in vivohemostatic agent or agents in these compositions.

The therapeutic use of activated PCC would be improved by purifying theeffective hemostatic agent. This would result in the administration ofless extraneous protein to the patient. In addition, the economics ofprotein fractionation would be enhanced by diverting the unwantedresidue from the purification to the manufacture of other therapeuticproducts.

Accordingly, it is an object of this invention to identify a hemostaticagent in activated PCC that can be isolated and used alone for thetherapy of blood clotting deficiencies.

It is a further object to identify such a hemostatic agent having a lowpotential for adverse thrombosis such as disseminated intravascularcoagulation.

It is an additional object to identify such an agent which can be usedso as to not stimulate patients to generate clotting factor inhibitors.

These and other objects of the invention will be apparent from thespecification as a whole.

SUMMARY OF THE INVENTION

Applicant has found that purified factor VIIa is a hemostatic agent inits own right. It does not require the administration of any otheractivated or unactivated factor to be therapeutically effective inachieving hemostasis. This was particularly surprising with respect tofactors IXa and Xa since the art has speculated frequently that thesefactors are likely candidates for the hemostatic effect of activatedPCC. The effectiveness of factor VIIa was also surprising because theexperimental animals were not deficient in factor VII and thereforecould presumably have generated all the factor VIIa that would have beenneeded to correct bleeding.

Accordingly, the method herein comprises administering to a patienthaving a clotting factor defect such as a deficiency or inhibitor aneffective hemostatic amount of a composition in which the soleeffective, activated hemostatic agent is factor VIIa.

The factor VIIa to be used in the method of this invention is free of aquantity of activated blood clotting factors which alone would besufficient to exert an in vivo, clinically hemostatic effect in theexperimental dog model described herein. Thus, it is conceivable thatthe compositions of this invention may contain activated blood clottingfactors other than factor VIIa. However, the activity of suchcontaminant factors will be so low that no clinically detectablehemostatic effect will be observed if the VIIa activity of thecontaminated composition is inhibited or physically separated from thecontaminants and the contaminants then administered under substantiallythe same conditions, e.g., infusion rate and concentration, as wouldhave been the case when factor VIIa was present in the contaminatedcomposition. Alternatively, the contaminated composition can be purifiedfree of factor VIIa, e.g. by electrophoresis, and the residue assayedfor clotting activity in a non-activated partial thromboplastin timetest or other general assay for endogenous clotting activity. If theclotting activity as measured by the Nemerson method before and afterseparation of factor VIIa has decreased more than three times over acontrol using pure factor VIIa then the composition is deemed to containa sufficiently low level of contaminant clotting proteases to enable itto be used in this invention.

Hemostatic effect or amount defined as the cessation of bleeding in astatistically significant number of hemophilia A dogs as described belowin Example 1 within about 30 minutes after the infusion of the testsample, which cessation was accompanied with an insignificant drop inhematocrit over a period of 3 hours following infusion.

Such compositions ordinarily will contain less than about 1 unit offactor Xa/ml and less than about 0.001 unit of thrombin/ml. Thedifference obtained by subtracting total factor IX activity from factorIX precursor activity, which yields an indirect measure of factor IXa,will be no more than about 15 units/ml and is generally less than about5 units/ml. The unit activities of these activated factors weredetermined by the methods set forth in copending U.S. patent applicationSer. No. 116,187; factor Xa was determined by the chromogenic methoddescribed therein.

It is not necessary that any contaminant activated clotting factors bephysically separated from the therapeutic composition since they may beinactivated or inhibited in situ. for example by inclusion ofantithrombin III and low levels of heparin, on the order of about 1 to 3units of heparin/μg factor VIIa. However, it is preferred to purify thefactor VIIa, either separating factor VII from residual unactivatedclotting factors and then activating, or by purifying factor VIIafollowing activation. Of the two alternatives, it is preferred to purifyfactor VII prior to activation. The separated clotting factors such asfactors IX and X are more versatile if the option to activate them stillremains. Additionally, the activation may be more readily controlled andthe quantity of starting material one must deal with is considerablyreduced if factor VII is purified prior to activation.

The above discussion has dealt with the presence of activated clottingfactors in the therapeutic composition. Unlike the case with activatedclotting factors, the unactivated precursor forms of such factors may bepresent in the compositions up to an amount which results in suchdilution of factor VIIa that administration of factor VIIa in atherapeutically effective dose would entail infusion of therapeuticallyunacceptable amounts of protein. This is quite a high concentration andas a practical matter imposes no limits in the case of typical plasmafractionation products. The amounts of unactivated factors II, VII, IXand X will typically range from about 1-10, 30-250, 0-30 and 1-30units/ml, respectively. Preferably, the compositions are essentiallyfree of factors IX and II but contain factors VII and/or X in the aboveconcentrations. The unit activities were determined by the methodsdescribed in the above-cited U.S. patent application.

Other blood plasma proteins than the activated or unactivated clottingfactors may be present in the compositions to be used in the inventionherein. Again, contamination with extraneous protein is preferablyavoided but is not critical. It is desirable, in fact, to leave someprotein in the purified factor VIIa to act as a stabilizer. For example,it is known that albumin stabilizes factor VIIa activity. Generally aprotein concentration greater than about 1 mg/ml is satisfactory toachieve factor VII stability although this amount is not critical.

Other physiologically-acceptable stabilizers may be included in thefactor VIIa-containing compositions to be used in accordance with thisinvention. Examples include non-reducing sugars, polysaccharides such aslow molecular weight dextrins, polyalcohols such as sorbitol orglycerol, amino acids including glycine, and antioxidants such asbisulfite or ascorbate. The stabilizers are generally present in aconcentration of about from 0.1 to 3% weight/volume.

Factor VIIa is useful in treating a variety of clotting factorinhibitors and deficiencies, in particular patients having deficienciesor inhibitors of factors VIII, IX and XI. Patients having inhibitors offactors VIII or IX have been observed. Inhibitors are substancesbelieved to be antibodies against the inhibited factors. Thesesubstances interact with the factors, probably by immune binding andsteric hindrance, so as to prevent their participation in the clottingmechanism. Inhibitors are a particularly acute problem because inhibitortiters generally increase in response to even larger doses of clottingfactor, and thus tend to eventually overcome high dosage therapeuticapproaches. This invention has particular applicability in the treatmentof factor VIII inhibitors.

Deficiency syndromes exist where the factor itself is lacking, ispresent in ineffectual concentrations, or is a protein having someantigenic similarities to the clotting moiety but which is substantiallynonfunctional in the clotting system. Deficiencies may result from avariety of causes, most commonly of congential origin or the destructionof clotting factor-producing organs. The most serious clotting factordeficiencies are those of factors VIII, IX and XI, and defects of theclotting mechanism attendant chronic liver disease such as alcoholiccirrhosis and viral hepatitis.

Factor VIIa is preferably administered upon diagnosis of a bleedingepisode rather than prophylactically. It should be administered as anaqueous solution by infusion rather than by bolus injection. Theactivity of factor VIIa in the infusion solution and the dosage offactor VIIa activity will necessarily depend upon the therapeuticcircumstances facing the clinician, e.g., severity and location of thebleed and the general condition of the patient.

The activity of factor VIIa in an infusion solution may vary over a widerange and is not critical, although the overall protein concentration inthe solution should be maintained to aid in stabilizing the factor.Generally, suitable infusions will contain about from 0.1 to 10.0 μg offactor VIIa/ml. Albumin or other stabilizing proteins should be presentin the infusion at about from 1 to 5 mg/ml.

Factor VIIa is preferably dosed as the mass of double chain moietypresent in the composition to be used. Methods are well known in the artfor measuring and distinguishing the mass of one protein in admixturewith other proteins. Such techniques include electrophoresis orimmunoassay.

A convenient shorthand modification of the mass dose technique is tosimply measure the factor VII content of a composition prior to takingsteps to activate the proenzyme. Then conditions are selected foractivation which have been determined previously with purified factorVII from the same source to yield the maximum increase in factor VIIaactivity (expressed factor VII activity). Since the molecular weights offactors VII and VIIa are substantially the same, the mass of factor VIIawill be substantially the same as the starting factor VII. And since itis a given that activation conditions have been maximized, it may beassumed that substantially all factor VII has been converted to factorVIIa.

Alternatively, dosage may be expressed in factor VII units. However, oneshould first take steps to ensure that the test material is free fromsubstantial contamination by factor VII and non-factor VIIa activatedclotting factors, which will interfere in the factor VII clotting assay.The material should be purified substantially free of factor VII becausefactors VII and VIIa both exhibit the same qualitative activity in theclotting mechanism, even though the activated form is about from 10 to100 times more active than factor VII. Since the assay is also aclotting assay, it too is incapable of distinguishing factor VII fromfactor VIIa. Also, since the method is based on the formation of afibrin clot activated factors such as factor Xa or thrombin, which cangenerate fibrin as well, will contribute to false positive results. Itis not critical to remove the factor VII in activated compositionsbecause factor VIIa is about from 10 to 100 times more active than theproenzyme; any contribution to the assay by factor VII will be swampedby the comparative vigor of factor VIIa in the assay.

The dosage of factor VIIa suitable for deficiency and inhibitor stateswill depend upon clinical circumstances such as the residual activity ofinhibited or deficient clotting factors, the site of the bleed and itsmagnitude, the general physical condition of the patient and a host ofother factors. Thus the dose must be varied by the clinician to arriveat the optimum therapy. This is of course a routine problem faced byphysicians in dealing with other therapeutic compositions and shouldtherefore be within the skill of the artisan. Generally a dose of aboutfrom 0.1 to 2.5 μg of VIIa per kg of body weight is preferred, withabout from 0.1 to 1.5 being most desirable. This dosage range willinduce hemostasis within about from 15 to 30 minutes, whereas untreatedsubjects generally continue to bleed uncontrollably. This range isintended only as a guide to the artisan and is not to be construed asappropriate for each and every therapeutic situation.

The frequency of dosage will depend upon the clinician's judgment aswell. Factor VIIa has a limited biological half-life in the bloodstream, so it is generally desirable to frequently infuse smaller doses.The frequency of dosing is ordinarily every three to five hours. Lessfrequent doses can be employed in treating minor bleeding sites, e.g.,small hematomas, while major bleeds such as traumatic wounds orsurgical-dental procedures should require more frequent infusions.

Suitable factor VIIa compositions for use herein are well known. Themethods of Kisiel et al., "Biochemistry" 16 (9): 4189-4193 (1977) orBroze and Majerus, op. cit. can be used to produce factor VIIa suitablefor use herein. The factor VIIa compositions produced by these methodscontain residual activating protease (factors Xa or XIIa) which shouldbe neutralized by separation from factor VIIa or by inhibition, forexample by addition of an excess titer of anti-activating factorantiserum. While the latter technique is workable, it is preferred topurify the factor VIIa, thereby avoiding the addition of foreign agentsto the composition.

Activating proteases may be separated from factor VIIa byinsolubilization either before or after the activation. It is preferredto insolubilize the proteases before activation by cross linking them toinsoluble, hydrophilic supports, e.g., cellulose, cross-linked dextransor nylon, using cross-linking agents such as carbodiimide or cyanogenbromide in accordance with techniques generally known in the enzymeinsolubilization art. Factor VIIa fails to exert any detectablefeed-back proteolytic activity on such immobilized proteases.

Alternatively, factor VIIa can be separated from the activating proteaseby generally known protein purification techniques, e.g.,electrophoresis, ultracentrifugation, immune affinity chromatography,ion exchange chromatography and the like.

The activation procedure of the foregoing authors may be furthermodified by the general procedure described in copending U.S. patentapplication Ser. No. 116,187. This procedure has as its foundationdetermining in advance of the completion of activation the conditionsneeded to achieve a factor VIIa composition of substantiallypredetermined activity. The method comprises

(a) providing a starting material comprising factors VII and IX;

(b) substantially separating factor VII from factor IX;

(c) activating the factor VII-enriched fraction from step (b) by

(i) selecting a condition which can be varied to control the degree offactor VII activation;

(ii) prior to the completion of activation, determining the magnitude ofthe condition needed to activate the factor VII to a predetermineddegree of activation;

(iii) setting the condition to said magnitude;

(iv) conducting the activation of the factor VII in accordance with thecondition; and

(v) terminating the activation at the predetermined degree ofactivation.

Generally only one condition of the activation is permitted to vary, andthis is usually the period of time that activation is allowed toproceed.

The magnitude of the selected condition is determined in one of twoways, or a combination of both. In the least preferred of the twomethods, the condition is determined by removing aliquots of thecomposition after activation has been commenced, terminating theactivation of each aliquot, determining the degree of activation of eachaliquot and calculating the magnitude of the condition necessary toachieve a predetermined degree of activation of the fraction.

Alternatively, the condition magnitude may be determined by removingaliquots of the composition prior to activation, varying the conditionamong the aliquots, activating the aliquots in accordance with thecondition set for each aliquot, terminating the activation, determiningthe degree of activation of each aliquot and calculating the magnitudeof the condition necessary to achieve a predetermined degree ofactivation of the composition. This embodiment has the advantage thatone cannot overrun the predetermined activation level, as could be doneduring the assay of aliquots withdrawn from a bulk lot which issimultaneously undergoing activation.

The degree of activation is generally monitored by following theincrease in factor VII activity to a desired percentage increase, e.g.,15 times the starting activity. The method of Seligsohn et al., "Blood"53 (5): 828-837 (1979) should be used for factor VII compositions whichcontain significant amounts of other clotting factors. This method isnot an absolute measure of factor VIIa. Instead, it assays the change inratio of factor VII to VIIa and is therefore a useful method formonitoring production of VIIa from the proenzyme.

EXAMPLE 1

The studies in this and the following examples were conducted on inbreddogs having hemophilia A. Hemostasis in such animals was followed byobserving bleeding or clot formation at a 5×2×1.5 mm wound (templatebiopsy) made surgically in the gingiva superior to the maxillary cuspid.In normal dogs, bleeding from the wound closed in 5±2 minutes and sealedtightly with a concave contour without rebleeding. In contrast,hemophilic dogs formed an abnormal convex clot over the wound and rebledfor several days if untreated. The hematocrit would drop by 2-10percentage points in association with uncontrolled bleeding.

For all infusions the template biopsy and a post transfusion biopsy fromthe posterior of the mouth were fixed in neutral buffered formalin,divided and embedded in either paraffin or a methacrylate plastic andafter sectioning stained for platelet plugs and for fibrin. Nomicrovascular thrombosis was detected in the sections reviewed. This isconsistent with the fact that no significant changes in either plateletcounts or fibrinogen were observed.

The factor VIIa used in these studies was prepared by the method ofKisiel et al., op cit. from human plasma rather than bovine. The factorXIIa used in the activation was inhibited by the addition of rabbitantiserum raised against human factor XII and having an anti-factor XIItiter of 50 μg. factor XII neutralized/ml antiserum by diisopropylphosphorofluoridate. The purified factor VII prior to activationcontained 3,250 units of factor VII/mg of protein (as measured by themethod of Nemerson and Clyne, "J. Lab. Clin. Med." 83:301 [1974].Benzamidine was removed from the factor VII by gel chromatography priorto activation. The factor increased the apparent factor VII activityapproximately 10 fold after activation with factor XIIa when assayedusing rabbit brain thromboplastin. VIIa thus generated was usually usedwithin two hours, but could be used successfully within 72 hours ifstored at 8° C. The concentration of factor VIIa was 240 μg/ml, and thisconcentration was used throughout the examples. The preparations used inExamples 1, 2 and 4-6 were believed to contain significant residualfactor VII.

A 13.6 kg hemophilic dog was selected, a biopsy was performed on day 1,and another on days 7 and 8. On day 1 the dog was infused withunactivated factor VII at a dose of 1.8 μg/kg. Generally, in thisexample and those that follow, infusions were made no longer than 30minutes after biopsy. Bleeding was not obvious clinically after thisinfusion but serial determinations of hemoglobin implied incompletehemostasis. On day 7 another biopsy was done, and on this occasion 60 μgof factor VIIa was infused, or a dose of 4.4 μg/kg. Serial hemoglobinlevels dropped in spite of this treatment, and clinically the bleedingcontinued. Therefore on day 8 a new biopsy was performed, and infusionwas performed with a smaller dose of factor VIIa, that is 24 μg or 1.8μg/kg. Serial hemoglobin assays on this date and clinical observationindicated cessation of bleeding with this dose of factor VIIa. Plateletcounts during these three days of observation remained stable, althoughfor unknown reasons the platelet counts on day 1 were higher than thoseon days 7 and 8.

EXAMPLE 2

This study was performed on a hemophilic dog with a weight of 14.1 kgs.On day 1 a biopsy was performed and the dog was injected with 24 μg offactor VIIa (1.7 μg/kg). This particular preparation of VIIa had notbeen treated with the anti-XII antibody and factor XIIa was thereforepresent. The bleeding did not cease either clinically or by serialdetermination of hematocrit. The antibody was added to the remainder ofthe preparation and the injection was repeated at 4:15 in the afternoon.Hemostasis then appeared to take place since the hematocrit was 37 onthe afternoon on day 1 and 38 on the morning of day 2. On day 2 thebiopsy was repeated and infusion of another 1.7 μg/kg of VIIa wasrepeated. Clinically the bleeding stopped within 10 minutes. Serialhematocrits were 31 and 32, and bleeding was clinically sporadic.Another 1.7 μg/kg of VIIa was given at 4 p.m. Very little bleedingappeared overnight and on day 3 the hematocrit was again 31%. A newbiopsy was performed on day 3 with free bleeding obtained. 48 μg offactor VIIa was then infused, or a dose of 3.4 μg/kg. The drop inhematocrit over 24 hours indicated that this higher dose of VIIa hadbeen less effective than the lower dose.

On day 4, clots were removed with gauze and all biopsy sites bled,especially the 24 hour wound. The dog was injected with 1.7 μg/kg offactor VIIa plus 100 microliters of standard rabbit brainthromboplastin. The injection was tolerated well, and the wounds begansealing at 10 minutes; however, observations and serial hematocritdeterminations indicated that bleeding was continuing. At 2:45 the dogwas injected with 0.85 μg/kg of factor VIIa. The bleeding appearedclinically to stop, and hemostasis appeared to remain stable through thestudy. Note however, that this injection was repeated at 9 p.m. on day4, at 8 a.m. on day 5, at 8 a.m. on day 6 and at 8:30 a.m. on day 7.

EXAMPLE 3

Template gingival biopsies were performed on a 15.0 kg hemophilic dog ondays 1, 2 and 3 under local anesthesia. Infusion on day 1 was with 0.16μg of human factor VIIa/kg. There was no adverse reaction, as indeedthere was no adverse reaction to any infusion with human factor VII orfactor VIIa regardless of prior exposure throughout the study. In thiscase hemostasis appeared to be accomplished within 15 minutes, butrebleeding occurred within three or four hours of the infusion.Hematocrit was 35% late on the first day and 31% on the morning of thesecond day indicating some overnight bleeding. Infusion on day 2 waswith 1.6 μg of factor VIIa/kg. Again, the bleeding appeared to stop, andon this occasion appeared to not recur until some time during theevening. The recurrence of bleeding was documented by a 24 hour drop inhematocrit from 31% to 25%. On day 3, infusion was performed withapproximately 14 μg of factor VIIa/kg. On this occasion bleeding did notappear to cease clinically. The 24 hour drop in hematocrit on thisoccasion was from 25% to 15%. It was unclear whether the factor XIIagiven along with the factor VIIa might not have been completelyneutralized and thus might have triggered fibrinolysis. On day 4 the 24hour wound was curetted and bleeding resumed. Bovine serum albumin wasgiven as a placebo without cessation of the bleeding. 51 units ofProplex® prothrombin complex/kg were infused and the bleeding appearedto recur in minor fashion overnight, although the 24 hour drop inhematocrit was only from 15-14%. On day 5, 48 units of factor VIIIconcentrate were infused or 32 units of factor VIII per kg. The bleedingceased immediately and did not recur.

There was no consistent change on any of the days tested in apparentfactor VIII as assayed either with factor VIII deficient caninesubstrate or factor VIII deficient human substrate. There was also noconsistent change in activated partial thromboplastin time. Theprothrombin time, however, did appear to change. On day 1 when 0.16μg/kg of factor VIIa were infused, the prothrombin time shortening wasstriking and was evident within 15 minutes. It also was maintained forthe entire day as was hemostasis. On the third day when even a largerdose was infused, the prothrombin time also shortened throughout theday, in spite of the fact that hemostasis was not achieved. Fibrinogenassays were also done on all samples, and showed no consistent change.

EXAMPLE 4

The next dog studied weighed 13.6 kgs. Biopsies were performed underlocal anesthesia on days 1, 2, 3 and 4 of the study. The dose of factorVIIa given on day 1 was 0.59 μg/kg. Clinically, the wound appearedsealed at 30 minutes, although the clot over the wound was convex andappeared fragile. Serial hematocrit determinations during the afternoonand the next morning revealed a hematocrit drop from only 38% to 36%.Serial particle counting with concomitant hemoglobin determinations alsodocumented that very little blood was lost. The particle counting alsoindicated no significant change in platelet count. Therefore, hemostasisappeared to be achieved on this particular occasion in this animal withthe dose at 0.59 μg/kg of factor VIIa. On day 2 the infusion was with0.59 μg of unactivated factor VII/kg. In this case the hematocritdropped from a beginning value of 36, to 33 at 4 hours and 31 at 6hours, and also to 28 on the following morning. Clinically there wasalso some bleeding during that time. Therefore, from these two days, itappeared that factor VIIa was hemostatically effective and factor VIIwas not. On day 3 the dog was again infused with 0.59 μg/kg of factorVIIa. The hematocrit prior to infusion was 28%, at 2 and 4 hours postinfusion it was 29%. The wounds were oozing, however, so another dose of0.59 μg/kg was given at 3 p.m. In spite of this the hematocrit droppedto 21% overnight. On day 4, infusion was with unactivated factor VII at1.18 μg/kg. The hematocrit dropped from 21 preinfusion to 19 two hourspost infusion to 16 five hours post infusion. At that point, the animalwas infused with two vials of factor VIII concentrate (482 u, 35 μ/kg)which appeared to control the bleeding. The hematocrit did not dropsignificantly over the next 24 hours, and the animal recovered withoutfurther treatment. As in previous experiments, the prothrombin timeexhibited a mild shortening but none of the other assays seemed tochange significantly. The prothrombin time shortening was seen witheither activated or unactivated factor VII, but in vitro assays haveimplied that factor VII may be undergoing slow activation on storage inthe absence of benzamidine even without the addition of factor XIIa.

EXAMPLE 5

The dog studied in this example had weighed 14.1 kgs. This dog underwentgingival biopsies on day 1 and day 2 of the study, and also on day 4.The beginning hematocrit was 54%. The injection on day 1 was with 1.42μg of unactivated factor VII/kg. Hemostasis appeared to occur within 5minutes, and was maintained for the first 30 minutes of observation;however, bleeding resumed at 3 p.m. and serial hematocrits indicated abeginning hematocrit of 54%, followed by a hematocrit of 52% at 4 hoursand 47% at 6 hours. The hematocrit at 24 hours was 41% so this dose offactor VII clearly did not stop bleeding. On day 2, infusion was withfactor VIIa (20 μg, 1.42 μg/kg). Again early hemostasis appeared therule by observation, but serial determination of hematocrit documented afall during the day from 41% to 31%, and a further fall to 26% by thenext morning. Unactivated human factor VII was given on the third day ina dose twice that on day 1, i.e. 40 μg, 2.84 μg/kg. Clinically thebleeding appeared to slow down, and the 24 hour drop in hematocrit wasonly from 26% to 22%. On day 4 a new biopsy was performed and a dose offactor VIIa was given (40 μg, 2.84 μg/kg). This did not producehemostasis, and the hematocrit dropped further to 16%. On this occasion,there was very little change in the prothrombin time when unactivatedfactor VII was used, but there was a significant drop when activatedfactor VII was used on days 2 and 4.

EXAMPLE 6

A 15.9 kg hemophilic dog had biopsies performed on days 1, 2, and 3.Infusion on day 1 was with 1.26 μg of factor VII/kg. Clinically thebleeding was intermittent, and by serial determinations of hemoglobinand hematocrit there was very little change in hemostasis. On day 2factor VIIa was given in the dose of 1.26 μg/kg. Hemostasis was prompt,but serial determination of hemoglobin and hematocrit suggested,however, a slow loss of blood over the 24 hour period. On day 3, a newbiopsy was performed and a placebo injection was given. This dog bled nofurther, and no additional infusions were administered.

It is concluded from the foregoing examples as a whole that factor VIIais therapeutically useful in the treatment of clotting defects. Whileblood loss as evidenced by a fall in the hematocrit did occur aftertreatment with factor VIIa in some cases, the slope of the curve of aplot of the reduction in hematocrit was adjudged to be significantlyless than that of control animals during the initial 3-6 hours afterinfusion.

I claim:
 1. A method for making a therapeutic composition, comprising(a)providing a starting material comprising factors VII and IX; (b)substantially separating factor VII from factor IX; (c) activating thefactor VII-enriched fraction from step (b) by(i) selecting a conditionwhich can be varied to control the degree of factor VII activation; (ii)prior to the completion of activation, determining the magnitude of thecondition needed to activate the factor VII to a predetermined degree ofactivation; (iii) setting the condition to said magnitude; (iv)conducting the activation of the factor VII in accordance with thecondition; and (v) terminating the activation at the predetermineddegree of activation.
 2. The method of claim 1 wherein the factor VII isactivated by factor XIIa.
 3. The method of claim 1 wherein the selectedcondition is the time of exposure of factor VII to factor XIIa.